Petroleum-derived fuels have served the mankind for ages. However, the recent awakening to the realization of dismal scenario of fossil fuel availability, perils of petroleum resource exhaustion, and stringent environmental legislation governing worldwide, has led to search for alternative energy sources (Herrera et al., 2004; Li et al 2010). Therefore, several alternative fuels are being investigated, which can either completely replace the petroleum derived fuels (gasoline and diesel) or can be blended with petroleum fuels to certain proportions, without requiring specially adopted engines in vehicles.
Amongst the other alternative fuels, Butanol was found to be of the best choice as it is superior replacement for gasoline, meeting the societal needs. Biobutanol is unequivocally a better fuel replacement for gasoline and is preferred in other industries for safety reasons. Being renewable, butanol helps in curbing the now so-called notorious “carbon” and other harmful emissions in the form of hydrocarbons; particulate matter; benzene, toluene, ethylbenzene, xylene (BTEX); and other undesirable elements (Sharma et al., 2010).
Butanol or butyl alcohol (sometimes also called biobutanol when produced biologically), is a primary alcohol with a 4 carbon structure and the molecular formula of C4H10O. It is primarily used as a solvent, as an intermediate in chemical synthesis, and as a fuel. Today, there is a paramount interest in producing fuels like butanol and ethanol using microorganisms by fermentation focusing on the environmental aspects and renewable nature of this mode of production. Butanol is a superior fuel and has more calorific value than ethanol (Qureshi and Blascher, 2000). Butanol has higher energy content (110,000 Btu's per gallon for butanol vs. 84,000 Btu per gallon for ethanol). It is six times less “evaporative” than ethanol and 13.5 times less evaporative than gasoline, can be shipped through existing fuel pipelines where ethanol must be transported via rail, barge or truck (Jones and ‘Woods, 1986).
Butanol is an important industrial solvent and potentially a better fuel extender than ethanol. Current butanol prices as a chemical are at $3.75 per gallon, with a worldwide market of 370 million gallons per year. The market demand is expected to increase dramatically if green butanol can be produced economically from low cost biomass. In addition to its usage as fuel, butanol can be used as a solvent for a wide variety of chemical and textile processes, in organic synthesis and as a chemical intermediate. It is also used as paint thinner and a solvent in other coating applications where it is used as a relatively slow evaporating latent solvent in lacquers and ambient-cured enamels. It finds other uses such as a component of hydraulic and brake fluids (Mutschlechner et al, 2000)/It is also used as a base for perfumes, but on its own has a highly alcoholic aroma.
Since the 1950s, most butanol in the United States is produced commercially from fossil fuels. The most common process starts with propene, which is run through an hydroformylation reaction to form butanal, which is then reduced with hydrogen to butanol. Butanol is produced by fermentation, from corn, grass, leaves, agricultural waste and other biomass.
Production of industrial butanol and acetone via fermentation, using Clostridium acetobutylicum, started in 1916. Chime Wizemann, a student of Louis Pasteur, isolated the microbe that made acetone. Up until the 1920s, acetone was the product sought, but for every pound of acetone fermented, two pounds of butanol were formed. A growing automotive paint industry turned the market around, and by 1927 butanol was primary and acetone became the byproduct.
The production of butanol by fermentation declined from the 1940s through the 1950s, mainly because the price of petrochemicals dropped below that of starch and sugar substrates such as corn and molasses. The labor intensive batch fermentation system's overhead combined with the low yields contributed to the situation. Fermentation-derived acetone and butanol production ceased in the late 1950s.
Acetone butanol ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations. It was ranked second only to ethanol fermentation by yeast in its scale of production, and is one of the largest biotechnological processes ever known. The actual fermentation, however, has been quite complicated and difficult to control. ABE fermentation has declined continuously since the 1950s, and almost all butanol is now produced via petrochemical routes. In a typical ABE fermentation, butyric, propionic, lactic and acetic acids are first produced by C. acetobutylicum, the culture pH drops and undergoes a metabolic “butterfly” shift, and butanol, acetone, isopropanol and ethanol are formed. In conventional ABE fermentations, the butanol yield from glucose is low, typically around 15 percent and rarely exceeding 25 percent.
The key problem associated with butanol production is butanol toxicity/inhibition of the fermenting microorganism, resulting in low butanol titer in the fermentation broth. (Ezeji et al, 2007). The production of butanol was limited by severe product inhibition. Butanol at a concentration of 1% can significantly inhibit cell growth and the fermentation process. Consequently, butanol concentration in conventional ABE fermentations is usually lower than 1.3 percent. Butanol is highly toxic to biological systems at quite low concentrations of 2% (Jones and Wood, 1986). This toxicity may be because butanol localizes in the plasma membrane and disrupts a number of physiological processes including membrane permeability, solute transport, and maintenance of proton motive force, conformation and activity of intrinsic membrane proteins. Efforts are being made to improve the butanol tolerance level in different species of Clostridia with varying degree of success (Evan and Wang, 1988). Recent interest in the production of butanol has led to re-examination of ABE fermentation, including strategies for reducing or eliminating butanol toxicity to the culture.
In the past 20+ years, there have been numerous engineering attempts to improve butanol production in ABE fermentation, including cell recycling and cell immobilization to increase cell density and reactor productivity and using extractive fermentation to minimize product inhibition. Despite many efforts, the best results ever obtained for ABE fermentations to date are still less. Optimizing the ABE fermentation process has long been a goal of the industry.
Butanol is currently produced worldwide at over 1.4 billion gal/year by chemical route. The market demand is expected to increase dramatically if butanol can be produced economically from low-cost biomass. Therefore, development of processes to produce biobutanol using renewable energy sources such as lignocellulosic crops is gaining impetus (Qureshi et al., 2010).
U.S. Pat. No. 4,521,516 provides a novel asporogenic strain of C. acetobutylicum produced by growing a spore-forming strain in a continuous culturing reactor. Culturing is conducted at a dilution rate which prevents accumulation of butanol and acetone in the medium. Culturing at this dilution rate is continued until the asporogenic strain is obtained.
U.S. Pat. No. 5,192,673 provide a biologically pure asporogenic mutant of C. acetobutylicum is produced by growing sporugenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which has been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents. However the fermentation herein includes three stage continous fermentation hereby making the process expensive and time consuming.
U.S. Pat. No. 6,358,717 provide a method of producing high levels of butanol using a fermentation process that employs a mutant strain of Clostridium beijerinckii. The mutant is a hyperamylolytic strain that is able to produce high titres of butanol in a glucose/starch rich medium. However there is no claim on the solvent tolerance of the strain.
U.S. Pat. No. 4,757,010 and European patent application EP 00111683 provides an improved strain of Clostridium for increased tolerance to butanol. JP03058782 provides Clostridium pasteurianum CA 101 stock (FERM P-10817) as a mutant of genus Clostridium bacterium having analog resistance to fermented intermediate of butanol and butanol producibility. U.S. Pat. No. 4,539,293 demonstrates the use of co-culture of microorganisms of the Clostridium genus, one favors the production of butyric acid and the other supports the formation of butanol. Japanese patent application J P 63157989 provides production of butanol by culturing a different strain Clostridium pasteurianum var. 1-53 (FERM P-9074) in a liquid medium containing a carbon source, a nitrogen source and other inorganic salts at 28-33 deg. C. under slightly acidic pH condition in anaerobic state for 2-4 days.
U.S. Pat. No. 4,777,135 describes a method of producing butanol by fermentation which comprises culturing under anaerobic conditions a butanol-producing microorganisms in a culture medium containing fluorocarbons. This process is not feasible on a commercial scale as the fluorocarbons are environmentally not safe
U.S. Pat. No. 4,605,620 provides a process for butanol by using a medium containing carbohydrate and phosphate, wherein the experiments were performed with a total phosphate content of 1.0-0.4 mmoles. This process poses a restriction wherein the phosphate limiting medium is required.
U.S. Pat. No. 4,560,658 provides a process for the production of butanol by fermentation of carbon containing compounds with C. acetobutylicum wherein the fermentation is conducted in an aqueous medium containing a sufficient concentration of dissolved carbon monoxide. However the use of carbon monoxide makes the process environmentally unsound.
U.S. Pat. No. 4,520,104 provides a process for the continuous production of butanol by fermentation of carbohydrates with C. acetobutylicum. This process combines continuous inoculum production at a high dilution rate and cycling the fermentation broth through material which adsorbs butanol whereby a vigorous cell population is maintained in the fermentation reactor for extended periods of time. The process is devised to remove the butanol produced in the broth so as to prevent its toxicity on the cells
Japanese patent JP 62278989 provides a fermentation process for the production of acetone and butanol, by keeping a butanol-producing strain in resting state, adding a carbon source to the cell to effect the production of acetone and butanol in a short time, recovering and concentrating the butanol-producing strain, subjecting to the heat shock and adding to a fermentation tank Heat shock is required in the process.—to activate the spores of Clostridium and is pretty routine.
Japanese patent application provides an anaerobic cellulolytic germ, e.g. Clostridium cellobioparum ATCC 15832 or Ruminococcus albus ATCC2721 1, and Clostridium saccharoperbutylacetonicum are inoculated into a culture medium containing a material containing cellulose, e.g. wood, waste paper or pulp, as a main carbon source, and cultivated at 25-45° C. and 4-9 pH under anaerobic conditions for about 2-20 days to collect the aimed compound, containing oxygen, and consisting essentially of butanol from the resultant culture. This process is time consuming and takes about 20 days for completion, hence not feasible on a large scale.
Japanese patent 63269988 discloses butanol fermentation wherein yeast is subjected to autodigestion in a fermentation tank and proliferated prior to the inoculation of butanol-producing strain. The space in the fermentation tank becomes anaerobic and the temperature increases by the proliferation of yeast to perform butanol fermentation. An inefficient autodigestion would lead to contamination of the broth by the yeast
US20050233031 provides a process for producing butanol which includes treating plant derived material to provide an aqueous liquor containing sugars in a fermentation process to produce a fermentation product. The process involves several steps and therefore cumbersome and tedious.
Japanese Patent JP 200535328801 provides a method for producing butanol in which a culture solution is prepared by using a formulation of the food residue with the Japanese distilled spirit lees and water and butanol fermentation is carried out in the culture solution. The use of Japanese distilled spirit is limited to the production experiments performed in Japan.
French patent FR2550222 provides a two stage process wherein a first stage of seeding with C. acetobutylicum and a second stage of seeding with yeast which produces ethanol, the second stage being commenced when the pH of the fermentation medium of the first stage has reached a minimum value. The invention applies in particular to the production of butanol, acetone and ethanol from sugar beet and Jerusalem artichoke juices.
Indian patent application 2544/MUM/2007 provides a process for production of high yields of butanol by Clostridium acetobutylicum ATCC 10132. The process can be completed in a shorter span of time, using batch process through manipulation of various process parameters. The process can also be used for biomass based production of butanol. The process requires commercial media whereas in the present invention acid pretreated feedstock is utilised along with the mutant strain of Clostridium acetobutylicum resulting in higher yields of butanol with enhanced butanol tolerance.
The commonly employed feedstocks as reported in the literature for biobutanol synthesis are corn (Zea mays), banana stems (Musa sapientum), jatropha (Jatropha curcas), and karanja (Pongamia pinnata) (Liang et al., 2010, Pfromm et al., 2010). However, the use of food crops like corn, sugarcane, etc. will lead to the food v/s fuel situation. In order to avoid this situation, it becomes imperative to explore the potential non-edible feedstocks and their suitability for biobutanol synthesis.
Among the many feedstocks available for biobutanol production, jatropha and karanja have been found to be most suitable due to their favourable attributes such as hardy nature, short gestation time of about 3 years, productive life of 50-100 years, not browsed by animals, adaptability to varied agro-climatic conditions and soil type, drought resistance and non competing with food crops for land and water sources.
The present invention provides the use of lignocellulosic biomass like jatropha seed cake and pongamia seed cake which is acid pretreated to disarray the cellulosic structure for extracting maximum reducing sugar. The hydrolysed sugar was used along with AnS medium components for biobutanol production.
The present invention also provides the use of the cellulose rich biomass i.e. banana stems which is acid pretreated to that release sugar which after hydrolysis produces biobutanol.
Although, there are reports where microbes have been exploited for the production of butanol by fermentation, there arises the need for an economically viable biosynthetic process for butanol production yet to be developed (Jesse et al, 2002).
The drawback associated with butanol production is butanol toxicity/inhibition of the fermenting microorganism, resulting in low butanol titer in the fermentation broth. Economic analysis has suggested that if butanol titers could be raised from 12 to 19 g/L, the separation cost could be cut down to half (Papoutsakis, 2008). Normally, the final titer of butanol in fermentation does not surpass 13 g/L because of feedback inhibition (Jones and Wood, 1986). Beyond this level, butanol is toxic to the bacterial cell and disrupts the membrane fluidity and function (Volherbst-Schneck et al., 1984).
In order to combat the effect of butanol toxicity, tolerant strains over express certain heat shock proteins (HSPs) like GroEL (Thomas et al., 2003, 2004) and alters its lipid composition by having more saturated fatty acids. This response known as ‘homeoviscous adaptation’, is believed to offset the physical changes caused by the environment and permits the cell to maintain its membrane at proper viscosity and surface ionic milieu for optimal cellular function (Baer et al., 1987).
According to the literature (Nishino and Yamaguchi, 2004), rhodamine 6G is P-glycoproteins substrate, mediates the energy-dependent efflux of certain toxic compounds from the bacterial cells. The existence of solvent efflux pumps in the cells can therefore be confirmed by rhodamine 6G accumulation in bacterial cells (Lazaroaie, 2009). The inventors of the present invention have used the above literature reference for assaying the solvent tolerant mutant strain and the wild type strain of C. acetobutylicum used for production of butanol.
Techniques such as gas stripping have been practiced to overcome the inhibitory effect of butanol produced during fermentation run (Ezeji et al., 2007). However, the process is plagued with inconsistent results and high energy expenditure. In the present invention, the inhibitory effect of butanol was overcome by improving the butanol tolerance of the microbe using chemical mutagenesis.
To combat the drawbacks of earlier inventions as cited in the prior art there arises the need for a process which yields enhanced production of butanol. In light of this the present invention has been focused on developing an ideal culture condition for the mutant strain of Clostridium which will result in enhanced butanol tolerance and subsequently the increase in yields of butanol.